Vascular grafts are in great demand because coronary artery diseases cause 12 million deaths in the world each year and account for half of all deaths in the United States. Despite the successful replacement of large-diameter blood vessels with non-biodegradable polymeric materials, critical issues remain in the creation of coronary heart disease-related small vascular grafts. So far no biomaterials and cel-based tissue- engineered blood vessel (TEBV) can meet the urgent needs of patients for coronary artery substitutes. The objective of this project is to develop a cell sheet engineering strategy potentially suitable for the production of a completely biological and mechanically strong off-the-shelf small-diameter vascular graft in large scale by manipulating human stem cells with biomimetic microenvironmental parameters. Our central hypothesis is that the well-defined biomimetic microenvironments composed of physiological-relevant low O2 concentration and hydrodynamic flows would effectively control the fate of human stem cells, enabling the reproducible production of a mechanically strong TEBV. Our goal is to engineer a small-diameter blood vessel that mimics the three-dimensional cellular organization in the natural blood vessel and can be used as allografts by any patient without time concerns. Aim I: Establish a robust off-the-shelf TEBV manufacturing scheme. Aim II: Evaluate the regeneration process and long-term patency of the TEBV in a rat abdominal model. Aim III: Evaluate the immune response of the TEBV as an allograft in an immune competent rat abdominal artery model. This research will establish a robust off-the shelf small-diameter TEBV fabrication strategy using stem cells. Upon completion, this novel cell sheet-based strategy will overcome the limitations of current biomaterial- and cell-based approaches to provide a completely biological small-diameter vascular graft that can sustain the high blood pressure, stimulate the functional vascular tissue regeneration, and induce integration by the host tissue in vivo.